This invention relates to gene transfer wherein a desired gene is delivered to a eukaryotic cell with applications for gene therapy. Such gene delivery may be accomplished in vivo, or may be accomplished in vitro, followed by the in vivo administration of such eukaryotic cells to a host. More particularly, this invention relates to liposomes and similar transfection vehicles which include an adeno-associated virus rep protein, adeno-associated virus ITRs, and DNA encoding a desired protein, polypeptide or genetic transcript, such as messenger RNA, antisense RNA, or a ribozyme.
Adeno-associated virus (or AAV) has the unique ability to target the integration of its DNA into a host cell genome in a non-random, locus-specific manner. This is in contrast to other viruses such as retroviruses which integrate at random positions in the host genome.
The left open reading frame of adeno-associated virus encodes the rep proteins. Two promoters located at map positions 5 and 19 (promoters p5 and p19, respectively) control expression of the four proteins derived from this ORF. Rep proteins Rep 78 and Rep 68 are produced from p5 promoted transcripts, and rep proteins Rep 52 and Rep 40 are produced from p19 promoted transcripts. It has been demonstrated in vitro that the p5 promoted rep proteins (rep 78 and Rep 68) bind to a defined region of human chromosome 19 at the integration locus for AAV provirus.
It is therefore an object of the present invention to employ AAV rep protein and the AAV ITRs as part of a gene delivery system for achieving targeted integration of foreign genes. Such targeted integration would provide a more effective and safer method of gene delivery. Other gene delivery techniques achieve low levels of integration, often require actively cycling cells as targets, and if integration occurs, it happens at random sites in the genome. Random integration poses the potential danger of inadvertent activation of a deleterious gene (such as a protooncogene) or inadvertent inactivation of an essential gene.
Many clinical gene therapy experiments or protocols also employ viral-based gene delivery systems. Such procedures pose the risk of contamination with potentially pathogenic wild-type virus, which is a significant safety concern. Also, these systems may result in significant host immune responses to transfected cells that express viral proteins on their surfaces.